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Humidity control and hydrophilic glue coating applied to mounted protein

crystals improves X-ray diffraction experiments

Article  in  Acta Crystallographica Section D Biological Crystallography · September 2013

DOI: 10.1107/S0907444913018027 · Source: PubMed

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Acta Cryst. (2013). D69, 1839–1849 doi:10.1107/S0907444913018027 1839

Acta Crystallographica Section D

BiologicalCrystallography

ISSN 0907-4449

Humidity control and hydrophilic glue coatingapplied to mounted protein crystals improves X-raydiffraction experiments

Seiki Baba, Takeshi Hoshino,

Len Ito and Takashi Kumasaka*

Structural Biology Group, Japan Synchrotron

Radiation Research Institute (JASRI/SPring-8),

1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan

Correspondence e-mail:

kumasaka@spring8.or.jp

Protein crystals are fragile, and it is sometimes difficult to find

conditions suitable for handling and cryocooling the crystals

before conducting X-ray diffraction experiments. To over-

come this issue, a protein crystal-mounting method has been

developed that involves a water-soluble polymer and

controlled humid air that can adjust the moisture content of

a mounted crystal. By coating crystals with polymer glue and

exposing them to controlled humid air, the crystals were stable

at room temperature and were cryocooled under optimized

humidity. Moreover, the glue-coated crystals reproducibly

showed gradual transformations of their lattice constants in

response to a change in humidity; thus, using this method, a

series of isomorphous crystals can be prepared. This technique

is valuable when working on fragile protein crystals, including

membrane proteins, and will also be useful for multi-crystal

data collection.

Received 7 May 2013

Accepted 29 June 2013

1. Introduction

Cryopreservation of protein crystals substantially reduces

radiation damage (Haas & Rossmann, 1970). However, simple

cryocooling of a crystal is accompanied by possible crystal

distortion caused by ice formation during the cryopreservation

process owing to the presence of large solvent channels in

protein crystals (Garman & Schneider, 1997). The most widely

used effective technique for preventing ice formation is to

replace the crystal mother liquor by an aqueous/organic

mixture solution (Petsko, 1975) using a cryoloop, a simple

crystal-mounting tool composed of low X-ray-absorption

materials (Teng, 1990; Garman & Schneider, 1997).

Many developments have improved this technique. To

improve the handling of samples or to reduce scattering noise

from mounting apparatuses, a loopless mount (Kitago et al.,

2005; Watanabe, 2006), a crystal catcher using an adhesive

(Kitatani et al., 2008) and a microfabricated polyimide film

(Thorne et al., 2003) have been developed as alternatives to

standard cryoloops. To eliminate or reduce the amount of

cryoprotectants that permeate the crystals and may sometimes

cause damage to them, a cryoprotectant-free procedure

has been achieved by maximizing the crystal-cooling rate

(Warkentin et al., 2006; Pellegrini et al., 2011), and a method

using a high-pressure environment (Kim et al., 2005) has also

been reported.

Meanwhile, crystal cooling is generally thought to introduce

slight changes into protein structures. Therefore, to study

temperature-sensitive structures experiments at both room

and cryogenic temperatures are necessary (Halle, 2004;

Dunlop et al., 2005; Fraser et al., 2011). A capillary-free

mounting system (Kiefersauer et al., 2000) and a humidity-

control device (Sanchez-Weatherby et al., 2009) are well suited

to conducting these experiments. However, these systems

were originally designed to improve the resolution of protein

crystals as a post-crystallization treatment, in which lattice

transformation is induced by variable controlled humidity that

results in the hydration or dehydration of crystals. Therefore,

to maximize the effect of humid air, protein crystals should

be exposed to air by removal of the mother liquor around the

protein crystal. Since crystals exposed to air are fragile owing

to their high sensitivity to environmental humidity changes,

the applications of this technique have been limited.

To improve the stability of crystals while maintaining the

applicability of capillary-free mounting, we examined crystal-

coating materials and found a suitable water-soluble polymer

solution: aqueous polyvinyl alcohol (PVA) glue. This

substance is widely used not only as a stationery glue and as a

laundry starch but also in biological applications: for instance,

as a cryopreservation reagent for embryos (Nowshari & Brem,

2000) and as a mounting medium for microscopic subjects such

as microbes (Omar et al., 1978). Although protein crystals

coated with this material showed only a small sensitivity to

humidity changes, the benefits of conferring high stability and

affinity to various fragile samples far outweigh this limitation.

In this report, we introduce this technique, the humid air and

glue-coating (HAG) crystal-mounting method, and demon-

strate its success with various protein crystals.

2. Materials and methods

2.1. Water-soluble polymers

As the water-soluble polymer, we used polyvinyl alcohol

(PVA) with average polymerization degrees ranging from

2000 to 4500 (PVA2000, PVA2400, PVA3500 and PVA4500).

PVA2000, PVA2400 and PVA4500 were supplied by Japan

VAM & POVAL Co. Ltd, Japan (product Nos. JP-20, JP-24

and JP-45, respectively). PVA3500 was purchased from Wako

Pure Chemical Industries Ltd (product No. 163-16355).

Aqueous solutions of PVA4500 [8%(w/w) and 12%(w/w)],

PVA3500 [13%(w/w)] and PVA2400 [15%(w/w)] were

dissolved in water by brief heating (363 K) and stirring.

For flash-cooling experiments, humidity conditions that

vitrify the PVA solutions themselves were examined by

X-ray scattering. Initial solutions of 8%(w/w) and 13%(w/w)

PVA4500 gave a powder diffraction pattern of hexagonal ice

after flash-cooling without any humidity control. Similarly,

glue exposed to air of 92.3% RH (relative humidity) produced

a powder diffraction pattern of cubic ice (Fig. 1a), but at

92.0% RH no ice ring was observed (Fig. 1b). Since the final

resultant concentrations of PVA are affected by vapour

pressure, i.e. atmospheric humidity (Hwang et al., 1998), it is

essential to control the humidity in order to prevent the glue

solution from forming ice. In contrast, the degree of poly-

merization (PVA2000, PVA2400, PVA3300 and PVA3500) did

not seem to affect the vitrification and glues adjusted to 92%

RH or below did not produce any ice rings after flash-cooling.

Thus, 8%(w/w) PVA4500 was used as the mounting glue in all

of the experiments if not otherwise specified.

2.2. The humidity-control apparatus

We developed a simple humidity-control apparatus

equipped with a two-flow humidity generator (Fig. 2a). Dry air

was supplied by an N2-gas generator (Iwatani Co. Ltd, Japan)

and an HOHMI YC-4R air compressor (Yaezaki Kuatsu

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1840 Baba et al. � New crystal-mounting method Acta Cryst. (2013). D69, 1839–1849

Figure 1Diffraction images of flash-cooled PVA glue. (a) 8%(w/w) PVA4500 exposed to air of 92.3% RH. (b) 8%(w/w) PVA4500 exposed to air of 92.0% RH.

Co. Ltd, Japan). Saturated-humidity air was produced by

bubbling the dry air in a water bath kept at 301 K and cooling

to room temperature (296 � 1 K with a temperature stability

of �0.3 K h�1) during transportation. Next, it was mixed with

the dry air and the total flow rate was brought to 3 l min�1 or

greater using two SEC-N series digital mass-flow controllers

(Horiba Ltd, Japan) controlled by a PC running Windows 7.

Although this handmade humidity generator was used in this

study, we confirmed that a modified version of the commercial

humidity generator HUM-1 (Rigaku Co., Japan), which can

produce humid air at a high flow

rate, also worked for this purpose.

The humidity-control gas was

transported to a humid air nozzle,

which was produced from a flex-

ible Teflon tube with an inner

diameter of 10 mm. The outlet

temperature and the relative

humidity were measured inside

the humid air nozzle using an

SHT71 humidity sensor

(Sensirion AG, Switzerland). This

humidity-control device can

control the humidity range

between 0.1 and 94.5% RH. Since

the humidity was regulated by the

mixture of gases, humidity change

took place at a rate of between

0.2 and 1% RH min�1.

2.3. Experimental protocols ofthe HAG method

2.3.1. Apparatus setup. The

humidity-control apparatus was

installed on the SPring-8 BL38B1

beamline equipped with a one-

axis rotation goniometer (Kohzu

Seiki Co. Ltd, Japan), ADSC

Quantum 210 or 315 CCD

detectors and a GN2 nitrogen

cryostream system (Rigaku Co.).

All of the following operations

were performed at room

temperature (296 � 18 K with

a temperature accuracy of

�0.3 K h�1). The humid air

nozzle of the apparatus was

placed under the sample (Fig. 2a)

and the humidity was adjusted;

typically, the initial humidity was

83% RH. The cryostream nozzle

was removed from the sample

position to avoid interference

with the humid air (Fig. 2a). In

the following steps performed at

room temperature, the intensity

of X-ray radiation should be minimized in order to reduce

radiation damage. In the case of our beamline, SPring-8

BL38B1, an aluminium plate with a thickness of 600–1000 mm

was used to attenuate the X-ray beam with the full photon flux

of 1.17 � 1011 photons s�1 to 1/10 to 1/40 at a wavelength of

1 A, respectively.

2.3.2. Glue preparation. An aqueous glue solution

consisting of 8%(w/w) PVA4500 was used except for certain

crystallization conditions. PVA interacts with ions, especially

multivalent anions such as tartrate, phosphate and sulfate ions,

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Acta Cryst. (2013). D69, 1839–1849 Baba et al. � New crystal-mounting method 1841

Figure 2Overview of the HAG method. (a) The humidity-control apparatus: 1, design of the apparatus; 2, the humidair nozzle and the sample; 3, the configuration of the two-flow humidity generator. The apparatus wasassembled from the following components: i, digital mass-flow controller; ii, temperature-controlled air-bubbling bottle (2 l); iii, air-cooling bottles (2 l and 500 ml); iv, air-mixing bottle (100 ml); v, humid airnozzle; vi, temperature and humidity sensor; vii, PC to control and monitor humidity through i and ii; viii,cryostream nozzle; ix, crystal sample. (b) Crystal-handling procedures: 1, applying the PVA glue to acryoloop; 2, picking up a crystal; 3, steeping the crystal to be coated by the glue. (c) A mounted crystal: 1,just before the crystal was placed on the apparatus; 2, after exposure to air of 73.9% RH.

and forms a physical hydrogel. Therefore, it is often difficult

to thoroughly coat the crystals owing to a varying degree of

hydrogel elasticity that depends on the concentration and the

valences of the ions. It is known that plasticizing reagents

such as glycerol, ethylene glycol and polyethylene glycol 400

prevent gel formation and that 5%(v/v) glycerol is sufficient to

reduce gel formation (Sakellariou et al., 1993). Thus, we also

examined three modified methods of sample handling for ionic

solutions. (i) The protein crystal was soaked in reservoir

solution supplemented with 5%(v/v) glycerol before trans-

ferring the crystal into the glue. (ii) Glycerol was added to the

crystallization solutions to a final concentration of 5%(v/v).

(iii) In the case of crystallization solutions containing a high

concentration of multivalent ions (e.g. greater than 1 M), the

glue was prepared with equal volumes of pure glycerol and the

aqueous PVA solution.

2.3.3. Crystal coating and mounting. A small amount of

the glue solution was applied to and spread over a crystal-

mounting loop (e.g. LithoLoops; Protein Wave Co.) with a

typical diameter larger than the crystal (labelled 1 in Fig. 2b).

A crystal was directly picked up from a crystallization droplet

by the glue-coated loop without removal of the mother liquor

surrounding the crystal (labelled 2 in Fig. 2b). The scooped

crystal was kept steady for a few seconds so that it could be

thoroughly coated and covered by the glue on the loop

(labelled 3 in Fig. 2b). Next, the loop was mounted on the

diffractometer and exposed to the humid air. This operation

should be completed within 10–20 s to minimize transpiration

of the crystalline moisture.

2.3.4. Searching for a suitable humidity condition. A

destructive hydration shock could be induced if the glue were

diluted by absorbing a large amount of the droplet solution in

the crystal pickup (Fig. 2c). To avoid this, a suitable humidity

level needs to be established immediately. The swelling of the

glue and consequent formation of crystalline ice at cryogenic

temperature is another concern. The protocol for coping with

these problems is described in Fig. 3.

To search for a suitable humidity condition, the crystal

quality was not only judged by visual inspection but was

also evaluated by obtaining diffraction images, which were

processed using the HKL-2000 suite (Otwinowski & Minor,

1997).

2.3.5. Determining the suitable humidity. The suitable

humidity and the extent of the safe humidity range vary from

one protein crystal to another. In this step, we roughly

determined the appropriate humidity range in which the

crystal did not suffer from any unrecoverable damage such as

fragmentation and dissolution. In most cases, these types of

damage were apparent from careful observation; signs of

chaps or cracks on the crystal surface and volume expansion of

the glue will lead to damage to the crystal. In some cases, we

also observed that the crystal surface melted in a hypotonic

condition.

We started the experiments with 83.0% RH humid air and

changed it in steps of �2–3% RH. When a crystal suffered

from unrecoverable damage in this step, the next crystal was

mounted with a new humidity condition selected from the best

apparent status from the previous experiments, as judged by

analytical and algorithmic determination of the optimal value

for a unimodal function against all measured data points.

2.3.6. Tuning the humidity. Optimization of the suitable

humidity was conducted by checking the crystal quality by

obtaining one diffraction image at each scanned humidity

point. Initially, a coarse optimization with steps of 1–2% RH

was performed and was followed by a finer humidity optimi-

zation with steps of 0.1–0.5% RH. To ensure sufficient time to

equilibrate water distribution in the whole system, the crystal

diffraction check was delayed for a few minutes after each

humidity change. If any irreversible damage, i.e. a sudden loss

of resolution, increasing mosaicity or splitting of spots, was

detected in the diffraction pattern, the crystal was replaced by

a new crystal and the experiment was continued from the last

humidity point.

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1842 Baba et al. � New crystal-mounting method Acta Cryst. (2013). D69, 1839–1849

Figure 3A flowchart summarizing the humidity-optimization strategy in the HAGmounting method.

Once the humidity had been optimized, it was kept constant

without any further change. Before proceeding to the next

step, the crystal was maintained at the optimum humidity for

15 min to complete water equilibration. Since lower humidity

was preferred in many crystals, the glue was dehydrated and

shrunk at optimum humidity (labelled 2 in Fig. 2c).

2.3.7. Flash-cooling. In order to perform cryogenic

experiments at 100 K, the humid air stream was replaced by

the Rigaku GN2 cryostream. Firstly, the shutter of the cryo-

stream was closed and its nozzle was returned to the sample

position. After the humid air nozzle had been removed, the

cryostream shutter was immediately opened. Although the

humidity range for successful cryocooling and the optimum

humidity depended on the sample (Table 1), we empirically

determined that the initial humidity for unknown samples

should be 83.0% RH. However, the number and the variety

of experiments conducted in the present study may not be

extensive enough to establish a standardized condition that

can apply to a wide range of different crystals.

2.4. Protein crystals

The following five types of crystals were prepared to vali-

date the HAG method.

2.4.1. Lysozyme. Hen egg-white lysozyme was obtained

from Seikagaku Co. (product No. 100940). Lysozyme crystals

were grown using hanging-drop vapour diffusion at 293 K.

Hanging drops were prepared by mixing 5 ml of 35 mg ml�1

protein solution in 50 mM sodium acetate buffer pH 4.5 with

an equal volume of a solution consisting of 1.6 M sodium

chloride, 50 mM sodium acetate buffer pH 4.5. The volume of

reservoir solution consisting of 1.0 M sodium chloride, 50 mM

sodium acetate buffer pH 4.5 was 500 ml per well. For the

series of experiments with the lysozyme crystals, we carefully

selected crystals by size from the same batch.

2.4.2. Hydrolase RsbQ. The bacterial hydrolase RsbQ from

Bacillus subtilis was expressed and purified as described

previously (Kaneko et al., 2005). The new form of RsbQ

crystals was grown using hanging-drop vapour diffusion at

293 K as follows: hanging drops were prepared by mixing 1 ml

of 12 mg ml�1 protein solution in 50 mM HEPES buffer pH

7.5 with an equal volume of a reservoir solution consisting

of 200 mM NaI, 20% PEG 3350, 1 mM reduced glutathione,

50 mM HEPES buffer pH 7.5. The volume of reservoir solu-

tion was 150 ml per well. Thin plate crystals initially appeared

with a thickness of a few micrometres and the microseeding

technique improved the thickness to approximately 100 mm.

We tried cryoprotectant solutions prepared using glycerol,

ethylene glycol, polyethylene glycol 400, 2-methyl-2,4-

pentanediol and trehalose at a concentration of 25%(w/v),

which was the lowest concentration to vitrify the mother

liquor. However, the crystals were mechanically very fragile

and sensitive to environmental changes. Indeed, the crystals

gradually delaminated and crumbled once the cover slides of

the hanging drops were opened.

2.4.3. Insulin. Bovine insulin was obtained from Sigma–

Aldrich Co. (product No. I550) and crystallized based on the

method reported by Gursky et al. (1992). The crystals of

insulin were grown using hanging-drop vapour diffusion at

293 K. Hanging drops were prepared by mixing 2 ml of

20 mg ml�1 protein solution in 20 mM sodium phosphate

buffer pH 12.0 with an equal volume of a reservoir solution

consisting of 1 mM EDTA, 0.4 M sodium phosphate buffer pH

10.4. The volume of reservoir solution was 500 ml per well. To

avoid PVA gel formation by the phosphate ion, the crystals

were soaked in reservoir solution containing 5%(v/v) glycerol.

2.4.4. BvRC. The bacterial membrane protein Blastochloris

viridis photoreaction centre (BvRC) crystal was provided by

Dr Takayuki Odahara. The crystals of BvRC were grown using

sitting-drop vapour diffusion at 288 K with a small glass vessel

for protein solution installed in a sealed plastic container.

Sitting drops were prepared using 400 ml of 18 mg ml�1 BvRC

solution in 2.5%(w/v) triethylamine phosphate, 2.5%(w/v)

1,2,3-hexanetriol, 0.3% lauryl dimethylamine oxide (LDAO),

1.51 M ammonium sulfate, 60 mM sodium phosphate buffer

pH 7.0. The volume of reservoir solution consisting of 5%(w/v)

triethylamine phosphate, 1.89 M ammonium sulfate, 100 mM

sodium phosphate buffer pH 7.0 was 12 ml in each setup. The

glue for the HAG mount was a mixture prepared from equal

volumes of pure glycerol and 8%(w/w) PVA4500 solution.

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Acta Cryst. (2013). D69, 1839–1849 Baba et al. � New crystal-mounting method 1843

Table 1Experimental conditions and diffraction data statistics for all of the examined crystals.

All X-ray diffraction data were collected at 100 K. Values in parentheses are for the highest resolution shell.

Hydrolase RsbQ Insulin BvRC LTC4S

Glue composition 13%(w/w) PVA3500 8%(w/w) PVA4500 4%(w/w) PVA4500 and50%(v/v) glycerol

4%(w/w) PVA4500 and50%(v/v) glycerol

Optimized humidity (% RH) 69.1 82.9 83.2 83.4Space group P212121 I213 P43212 F23Unit-cell parameters (A) a = 42.30, b = 43.05, c = 256.98 a = 77.74 a = 220.58, c = 113.26 a = 167.52Mosaicity (�) 0.33 0.44 0.33 0.30Resolution (A) 50.00–1.40 (1.42–1.40) 50.00–1.53 (1.56–1.53) 50.00–1.85 (1.92–1.85) 50.00–2.70 (2.80–2.70)Multiplicity 14.0 (14.3) 21.8 (21.7) 10.1 (7.8) 11.3 (7.5)Completeness (%) 96.8 (73.9) 99.9 (100.0) 99.8 (99.4) 100.0 (100.0)Rmerge† (%) 5.8 (42.8) 4.5 (38.7) 6.1 (49.7) 13.1 (49.5)Average I/�(I) 52.5 (4.0) 96.0 (12.9) 58.4 (4.3) 53.3 (5.5)

† Rmerge =P

hkl

Pi jIiðhklÞ � hIðhklÞij=

Phkl

Pi IiðhklÞ, where Ii(hkl) is the observed intensity of the ith measurement of reflection hkl and hI(hkl)i is the mean intensity of reflection hkl

calculated after scaling.

2.4.5. LTC4S. The human membrane protein leukotriene C4

synthase (LTC4S) was provided by Dr Hideo Ago (Ago et al.,

2007; Saino et al., 2011). The crystals of LTC4S were grown

using sitting-drop vapour diffusion at 293 K in a 96-well

crystallization plate. Drops were prepared by mixing 5–10 ml

of 6 mg ml�1 LTC4S solution in 20 mM MES–NaOH pH

6.5, 5 mM glutathione, 0.04%(w/v) n-dodecyl-�-d-maltoside

(DDM) with an equal volume of reservoir solution consisting

of 0.04%(w/v) DDM, 5 mM glutathione, 1.6–1.7 M ammonium

sulfate, 0.5–0.8 M magnesium chloride, 20 mM MES–NaOH

buffer pH 6.5. The crystals of LTC4S were transferred over-

night into harvesting solution consisting of 2.4 M ammonium

sulfate, 50 mM glutathione, 20 mM MES–NaOH buffer pH 6.5

(Saino et al., 2011). The glue used was the same as that used for

BvRC.

3. Results

3.1. The HAG method works with different types of crystals,including those of two membrane proteins

We tested this method on several protein crystal samples

following the procedure shown in Fig. 3 and described in x2.

Using this method, most of the samples successfully under-

went humidity-optimization experiments at room temperature

followed by full data collection at 100 K (Table 1). The most

notable result was obtained with the crystals of bacterial

hydrolase RsbQ from B. subtilis. The crystals were mechani-

cally fragile and were damaged by all of the soaking experi-

ments, in which split spots were observed in diffraction images

after flash-cooling (Fig. 4a). By employing an HAG mount

using 13%(w/w) PVA3500 glue without any other cryopro-

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1844 Baba et al. � New crystal-mounting method Acta Cryst. (2013). D69, 1839–1849

Figure 4Diffraction images of bacterial hydrolase RsbQ crystals. (a) A diffraction image from a crystal treated using a typical cryoprotection procedure. Thecrystal was mounted with a cryosolution composed of the reservoir solution supplemented with 25%(v/v) glycerol and then flash-cooled. (b) Adiffraction image from a crystal mounted with the HAG method. The crystal was mounted using 13%(w/w) PVA3500 glue with a humidity of 69.1% RHand then flash-cooled. Both samples were kept at 100 K.

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Acta Cryst. (2013). D69, 1839–1849 Baba et al. � New crystal-mounting method 1845

Figure 5Reversible crystal lattice transformation of glue-coated lysozyme crystals subjected to humidity changes. (a–c) The effects observed on thecrystallographic a axis (top), c axis (middle) and mosaicity (bottom) caused by humidity changes. (a) The response to a reduction in humidity. The filledcircles (solid line) indicate continuous measurements of a crystal treated with humid air starting at 87.7% RH and gradually decreasing to 67.6% RH.(b, c) The response to two-way changes in humidity. The lattice-parameter changes during the humidity-reduction process are plotted as filled circles. (b)Humidity reduction from 88.3 to 67.9% RH. (c) Humidity reduction from 87.1 to 69.8% RH. In (b) and (c) the triangles (dotted line) indicate the trace oflattice-parameter changes during the humidity-recovery process up to 92.5% RH (b) and 90.5% RH (c) after the previous humidity reduction. Inaddition, the lattice constants observed after flash-cooling are overlaid on (b). For flash-cooling, five new crystals were prepared at five differenthumidities (74.3, 79.5, 82.7, 87.3 and 88.6% RH). Parameters obtained from the experiments conducted at room temperature and 100 K are plotted asopen and filled squares, respectively. (d) Diffraction images collected at the humidity conditions indicated by the four wedges shown in (a).

tectants and an optimized humidity of 69.1% RH, cryopro-

tection of the RsbQ crystals was successfully achieved and a

full diffraction data set was collected (Fig. 4b and Table 1). In a

previous study with another crystal form, this enzyme showed

some structural changes induced by the binding of propylene

glycol as a cryoprotectant to the active site (Kaneko et al.,

2005). Therefore, it seems reasonable that the new crystal

form was sensitive to these permeable cryoprotectants.

However, the HAG method initially failed when we tested

crystals containing high concentrations of multivalent ions. It

is known that the PVA glue immediately reacts with the ions

to form a hydrogel. The stickiness of the gel made it difficult

to thoroughly coat the crystals with the polymer glue, as the

crystals stuck to the gel surface. To address this issue, we

added glycerol, a plasticization reagent for PVA, which

reduced gel formation (Sakellariou et al., 1993). The insulin

crystals usually required a glycerol concentration of 25%(v/v)

or higher as a permeable cryoprotectant in the conventional

cryocooling method, whereas they could be mounted and

cryocooled with only 5%(v/v) glycerol in the HAG method.

In this case the crystal was first soaked in reservoir solution

supplemented with 5%(v/v) glycerol to replace the mother

liquor and was then used in HAG mounting. The addition of

only 5%(v/v) glycerol was sufficient to prevent gel formation

by the 0.4 M phosphate ion contained in the mother liquor. By

contrast, for both BvRC and LTC4S crystals the addition of

5%(v/v) glycerol was insufficient to prevent gel formation

because both of the crystallization conditions contained high

concentrations of sulfate ions (1.9–2.5 M). However, addition

of glycerol to the polymer glue was effective for both crystals.

The glue prepared by mixing equal volumes of glycerol and

8%(w/w) PVA4500, i.e. a final concentration of 50%(v/v)

glycerol and 4%(w/w) PVA, was successfully subjected to

cryocooling without ice formation (Table 1). In the conven-

tional cryocooling method, a BvRC crystal soaked in cryo-

protectant containing 30%(v/v) glycerol was damaged in only

2 min (Baxter et al., 2005). However, the PVA glue and

glycerol mixture preserved the crystals for over 20 min during

a diffraction check at room temperature when mounted using

the HAG method.

3.2. Humidity change causes reversible latticetransformation of lysozyme crystals at room temperature

In addition to the cryoprotection effects, we also investi-

gated the transformation of lysozyme crystal lattices in

response to humidity changes. Firstly, we estimated the

humidity ranges in which glue-coated crystals remain stable.

Tetragonal crystals of hen egg-white lysozyme were mounted

by the HAG method without any additional cryoprotectants

and the humidity was reduced from 87.7% RH to the cata-

strophic point at which the crystal was damaged (Figs. 5a and

5d). During the dehydration process to 68.3% RH (Fig. 5a), a

0.73% shrinkage of the crystallographic a axis and a 1.45%

stretching of the c axis were simultaneously observed. With

further dehydration from 68.3 to 67.6% RH the crystal

suddenly cracked and the mosaicity surged (Figs. 5a and 5d).

Rehydration with increased humidity did not lead to recovery

of the crystal from cracking.

The reversibility of the response to dehydration was esti-

mated in the next two experiments, in which the humidity was

reduced from 88.3 to 68.1% RH immediately before the

catastrophic point and was returned to 92.5% RH (Figs. 5b

and 5c). The dehydration process gradually led to lattice

transformation along the a and c axes to 78.51 and 38.35 A,

respectively. Moreover, during the following rehydration

process the lattice parameters inversely traced the transitions

that were observed in the dehydration process. This showed

a linear relationship between the humidity and the lattice

constants, although a small nonlinear response and increased

mosaicity were observed in the first dehydration steps. Such a

nonlinear response was also observed by exposing a crystal to

constant humidity (Fig. 6). It typically took 20–30 min for the

moisture to reach equilibrium between the humid air and the

mounted sample, which initially contained excess moisture

derived from the glue. However, once the excess moisture had

been removed the crystal responded uniformly to a change in

humidity and was safely maintained over 2 h in the experiment

that involved successive dehydration, rehydration and re-

dehydration (Fig. 7).

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1846 Baba et al. � New crystal-mounting method Acta Cryst. (2013). D69, 1839–1849

Figure 6Time course of the lattice transformation of a crystal subjected to aconstant humidity of 80.4% RH plotted for the crystallographic a axis(top), c axis (middle) and mosaicity (bottom).

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Acta Cryst. (2013). D69, 1839–1849 Baba et al. � New crystal-mounting method 1847

3.3. Humidity affects the diffraction data quality offlash-cooled crystals

The effect of the HAG mount for cryocooling was examined

using five lysozyme crystals, each with a different humidity

condition, in which no ice-ring patterns were observed but for

which the mosaicities increased after cryocooling (Table 2 and

Fig. 5b). In particular, the samples exposed to high humidity

(87.3–88.6% RH) showed higher mosaicity (0.55–0.66�) than

the others (<0.46�). In terms of the resolution, the data set

with the highest resolution was obtained from the 82.7% RH

condition, which is in the middle of the tested humidity range

(Table 2). Both extremes in humidity may possibly induce

hypertonic or hypotonic stress of the crystals. As well as

desiccation, we also observed that a lysozyme crystal was

cracked by exposure to a high humidity of around 90% RH for

5 min or longer. In addition, there was an alternative limita-

tion in the cryogenic experiments; i.e. a solution of only PVA

treated with humidity above 92% RH gave ice-ring diffraction

on cryocooling. It has been reported that the water vapour

pressure of aqueous PVA (molecular mass 88 kDa) at

concentrations of about 60% or below remains approximately

at the saturated pressure of water vapour at 304.15 K (Hwang

et al., 1998). Thus, the PVA concentration might reach about

60%(w/v) by exposure to unsaturated humid air, leading to

successful vitrification.

4. Discussion

Our results demonstrate the high feasibility of the HAG

method for crystal mounting and cryocooling. We speculate

upon several possible reasons for the feasibility and efficacy of

this method.

(i) The nature of retaining moisture and buffering moisture

exchange. The glue prevents direct exchange of water vapour

between the crystal and the atmosphere. Instead, the glue

releases its own moisture, rather than that of the crystal, to the

dry atmosphere during crystal handling or humidity changes.

This moderate moisture-exchange system protects fragile

crystals from a hydration or dehydration shock. In addition,

the hypotonic property of the polymer solution retains the

moisture of a crystal. The osmotic pressure of the glue solution

depends on the concentration of its solute and is also affected

by the polymerization degree of the solute polymer according

to the Flory–Huggins theory; a longer polymer gives a lower

osmotic pressure at a given vapour pressure. Indeed, the

osmolality of the glue is almost negligible (<10 mmol kg�1)

compared with that of the crystallization solution of lysozyme

(osmolality >2000 mmol kg�1).

Figure 7Long-term stability accompanied by reproducible lattice-constant transitions of glue-coated lysozyme crystals treated with consecutive dehydration,rehydration and re-dehydration processes. (a) Time course of the transformation of a crystal subjected to the following programmed humidity changes.In the first stage of the program, the relative humidity was kept at 87.4% RH for 10 min after the lysozyme crystal had been mounted. Next, dehydration(87.4 to 70.0% RH) and subsequent hydration (70.0 to 90.1% RH) experiments were conducted with a stepwise humidity change at a rate of 2.5% RHper 5 min. Finally, re-dehydration (90.1 to 70.3% RH) was applied with a humidity-change rate of 5% RH per 10 min. (b) The reversible changes in thelattice constants in response to humidity changes. The relative changes of the crystallographic a axis (�a/a) and c axis (�c/c) and of the unit-cell volume(�V/V) are plotted with solid, dotted and grey bold lines, respectively.

(ii) High viscosity and slow diffusion. The high viscosity of

the glue guards the crystals from mechanical stress and slows

the diffusion of solvents, solutes and PVA itself. The high

viscosity may retard changes in the chemical environment

surrounding a crystal. The effects might also mitigate osmotic

shocks (Sugiyama et al., 2012).

(iii) Affinity for crystallization solutes. The glue might

provide a similar chemical condition to the crystallization drop

by absorbing the crystallization solution during crystal pickup.

Even in dried conditions, the absorbed chemicals might be

trapped by the glue through interaction with the hydroxyl

groups of PVA, preventing the condensation of chemicals

inside the crystal.

(iv) Suppression of ice crystals. Similarly to permeable

cryoprotectants, the hydrophilic polymer suppresses the

formation of ice crystals in the solution around the crystals.

Moreover, the hydrophilic glue can absorb the excess crys-

tallization solvent that usually contributes to ice formation.

In this sense, the use of the polymer glue is different from oil

coating, in which case a small amount of the excess solvent

may be left at the crystal–oil border.

Through the humidity-control experiments, the glue proved

to be suitable for fixing protein crystals on a cryoloop (Fig. 2c).

However, glue-coated lysozyme crystals showed a different

response to that of the uncoated crystals to humidity changes

(Dobrianov et al., 2001; Salunke et al., 1985; Kodandapani et

al., 1990; Kiefersauer et al., 2000), in which a large change in

lattice constants (maximally 10%) and hysteretic responses

were observed. Instead, our results are similar to the obser-

vation reported in soaking experiments with a higher

concentration of sodium chloride (Lopez-Jaramillo et al.,

2002); that is, the response was small (less than a 1% change

in lattice constants), the c axis expanded after dehydration

and a hysteretic response was not observed. The response to

humidity changes was reproducible regardless of the crystal

orientation, which is different from the experiments with the

uncoated crystals. These responses of the crystals in the HAG

method were rather subtle and the mosaicity was almost

constant. Therefore, this process might be regarded as an

elastic deformation induced by a smaller stress derived from

osmotic pressure.

However, a mosaicity change was often observed in the

initial steps of the mounting procedure (Figs. 5 and 6).

Because a relatively high mosaicity was reduced by dehydra-

tion in these cases, some part of this change might be caused

by the hypotonic effect of the glue on the crystals owing to

excess moisture in the glue or inappropriate initial humidity.

Therefore, this undesirable effect should be further mitigated

by adjustment of the osmolality of the glue and optimization

of the initial humidity.

Meanwhile, the mechanism that causes the catastrophic

damage upon dehydration remains unclear (Fig. 5a). In the

hypertonic condition, the polymer concentration exponen-

tially correlates with the osmotic pressure. In addition to the

pressure, mechanical force induced by dehydration of the

PVA hydrogel and salt displacement might cause significant

stress to crystals. In some cases, the glue surface became lumpy

and salt crystals appeared during the dehydration processes.

The surface roughness of the glue might reflect the formation

of PVA hydrogel, in which physical networks of polymer

molecules are formed by hydrogen bonds and ionic inter-

actions together with polar or ionic solutes. To avoid unre-

coverable damage, it might be necessary to optimize the

humidity and the amount of the plasticizing reagents.

The controllability of the osmotic pressure will aid crystallo-

graphers to manage lattice constants and isomorphism when

dealing with a series of crystals at room temperature. This

feature might be useful for multi-crystal data collection using

synchrotrons or X-ray free-electron lasers (Kern et al., 2012).

Since the flash-cooling process is not completely under our

control, the HAG method does not assure isomorphism

in cryocooled crystals but only provides fixation of initial

conditions for cryocooling. Thus, hierarchical cluster analysis

will be helpful for this purpose (Giordano et al., 2012).

Moreover, the hydrophilic glue will not work for preventing

ice formation in solvent channels of protein crystals, similar to

immiscible oils used for cryoprotection (Kwong & Liu, 1999).

Recently, multicomponent mixtures of cryoprotectants have

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1848 Baba et al. � New crystal-mounting method Acta Cryst. (2013). D69, 1839–1849

Table 2Experimental conditions and diffraction data statistics of lysozyme crystals.

Values in parentheses are for the highest resolution shell. The maximum resolution of each data set was determined as that where the CC1/2 (Karplus & Diederichs,2012) was around 0.8 in the highest resolution shell. The values of CC1/2 were calculated with the PHENIX package (Adams et al., 2010).

Optimized humidity (% RH) 74.3 79.5 82.7 87.3 88.6

Unit-cell parameters (A)a 78.39 78.27 78.38 78.39 78.49c 37.26 37.22 37.10 37.17 37.17

Mosaicity before/after cryocooling (�) 0.23/0.43 0.081/0.46 0.14/0.34 0.12/0.55 0.19/0.66Resolution (A) 50.00–1.18 (1.20–1.18) 50.00–1.19 (1.21–1.19) 50.00–1.13 (1.15–1.13) 50.00–1.15 (1.17–1.15) 50.00–1.15 (1.17–1.15)Multiplicity 6.3 (3.5) 6.6 (6.5) 6.9 (6.8) 6.6 (6.5) 6.8 (6.6)Completeness (%) 97.0(75.9) 99.7 (100.0) 99.9 (99.8) 99.9 (99.8) 98.7 (99.6)Rmerge† (%) 4.6 (49.5) 4.5 (67.1) 4.6 (65.9) 5.1 (65.8) 6.0 (81.5)Average I/�(I) 24.1 (2.8) 19.5 (2.6) 20.9 (2.7) 19.3 (2.6) 18.0 (2.3)CC1/2 0.999 (0.814) 0.999 (0.806) 0.999 (0.796) 0.999 (0.810) 0.999 (0.807)

† Rmerge =P

hkl

Pi jIiðhklÞ � hIðhklÞij=

Phkl

Pi IiðhklÞ, where Ii(hkl) is the observed intensity of the ith measurement of reflection hkl and hI(hkl)i is the mean intensity of reflection hkl

calculated after scaling.

successfully provided extended stabilization during cryopro-

tection to allow longer soaking periods without crystal cracking

or dissolving (Vera & Stura, 2013). Thus, combinatory use of

multicomponent permeable cryoprotectants such as polyols

and cryosalts would increase the flexibility of the method.

5. Conclusions

The new crystal-mounting method, the HAG method, is an

effective way of handling fragile protein crystals and is

compatible with experiments at both room and cryogenic

temperatures. Furthermore, treatment of samples with proper

humidity could produce desired lattice constants, potentially

resulting in improved isomorphism for multi-crystal data

collection using a series of crystals. This feature is required

for experiments with radiation-sensitive crystals or when

conducting experiments at room temperature using a

synchrotron or an X-ray free-electron laser X-ray source. We

are in the process of further improvement of the method.

We would like to thank Hideo Ago (RIKEN SPring-8

Center) and Takayuki Odahara (National Institute of

Advanced Industrial Science and Technology) for providing

the LTC4S and BvRC crystals, respectively. We also thank

Toshifumi Matsuoka and Yoshihiro Kimura (Japan VAM &

POVAL Co. Ltd) for providing various types of PVA materials

and Nobuhiro Tanaka (Rigaku Co.) for the modification of the

HUM-1 humidity generator. The authors are also grateful to

Tomonori Kaneko (University of Western Ontario) for critical

reading of the manuscript. This work was supported in part

by KAKENHI (23121537) to TK. All diffraction experiments

were performed on the BL38B1 beamline at SPring-8 with

the approval of the Japan Synchrotron Radiation Research

Institute (JASRI; proposal Nos. 2009B2091, 2010A1974,

2010B2012, 2011A2043, 2011A2054, 2011B2104, 2012A1834,

2012B1910 and 2013A1872).

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